title: Reproducible high yields of recombinant adeno-associated virus produced using invertebrate cells in 0.02- to 200-liter cultures.
abstract: The large amounts of recombinant adeno-associated virus (rAAV) vector needed for clinical trials and eventual commercialization require robust, economical, reproducible, and scalable production processes compatible with current good manufacturing practice. rAAV produced using baculovirus and insect cells satisfies these conditions; however, recovering rAAV particles from 200-liter bioreactors is more complicated than bench-scale vector preparations. Using a variety of processing media, we developed a reliable and routine downstream procedure for rAAV production that is scalable from 0.02- to 200-liter cultures. To facilitate the upstream process, we adapted the titerless infected-cell preservation and scale-up process for rAAV production. Single-use aliquots of cryopreserved baculovirus-infected insect cells (BIIC) are thawed and added to the suspension culture to achieve the desired ratio of BIIC to rAAV-producer cells. By using conditions established with small-scale cultures, rAAV was produced in larger volume cultures. Strikingly consistent rAAV yields were attained in cultures ranging from 10 liters to 200 liters. Based on the final yield, each cell produced 18,000 ± 6,800 particles of purified rAAV in 10-, 20-, 100-, and 200-liter cultures. Thus, with an average cell density of 4.32 × 10(6) cells/ml, ≥ 10(16) purified rAAV particles are produced from 100 to 200 liters. The downstream process resulted in about 20% recovery estimated from comparing the quantities of capsid protein antigen in the crude bioreactor material and in the final, purified product. The ease and reproducibility of rAAV production in 200-liter bioreactors suggest that the limit has not been reached, and 500-liter productions are planned.
authors.affiliation: Laboratory of Molecular Virology and Gene Therapy, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Cecchini
authors.forename: Tamas
authors.initials: T
authors.lastname: Virag
authors.forename: Robert M
authors.initials: RM
authors.lastname: Kotin
mesh_terms: D000818:Animals; D016367:Baculoviridae; D019149:Bioreactors; D002452:Cell Count; D018929:Cell Culture Techniques; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors; D007448:Invertebrates; D015203:Reproducibility of Results